PT91785A - PROCESS FOR THE PREPARATION OF POLIOXOMETALATES WITH ANTI-VIRAL ACTION AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM - Google Patents

Description

The present invention relates to a process for the preparation of anti-viral agents and pharmaceutical compositions containing polyoxyalkylated compounds of the formula: ## STR1 ## in which R 1 is H ,;

Background of the Invention

Field of the Invention: The present invention relates to certain polyoxyalkylate compounds, particularly polyoxometalate amino acid salts which are antiviral agents. In particular, such agents may be used for the treatment of retroviral infections such as HIV [HTLV-nr / LAV1, which cause acquired immune deficiency syndrome [AIDS]. The present invention also relates to a method of treatment of viral diseases such as AIDS which involves treating an HIV infected animal with a composition containing one or more of the compounds of the present invention. Other retroviruses may also be treated with the compounds of the present invention,

Discussion of prior knowledge The first time AIDS was detected in 1979, the number of cases reported by the Centers for Disease Control (CDCJ) has increased dramatically each year since then, and in 1982 CDC declared AIDS a new epidemic, SÕ in 1984, 3,000 new cases were declared,

On June 1, 1988, the World Health Organization (WHOJ announced that around 100,000 cases of AIDS had been registered worldwide. However, in fact, WHO estimated an estimated 200,000 cases worldwide. it was estimated that approximately 5,000,000 people were currently infected with HIV and at least 1,000,000 new cases of AIDS could be expected over the next 5 years.

In the United States, about 70,000 cases of AIDS have been reported by the U.S. Centers for Disease Control (CDC), only in 1987 were recorded close to 28 000, It was predicted that in the States t

United States will be registered between 365,000 and 38,000 total cases in 1992. In CDC it is estimated that 1 million or 1.5 million Americans are currently infected with HIV. It is evident that the cost of the AIDS epidemic in terms of human lives is doubtful and that the worst is yet to come.

The proposed causative agents of AIDS were retroviruses. Two of these retroviruses that are known to cause AIDS have been identified and isolated: LAV (Virus associated with lymph denatopathy and HTLV-III [Human T-cell leukemia virus]). Later, it was determined that LAV and HTLV-III are identical. The term human immunodeficiency virus type 1 (HIV 1) was presently considered to be the preferred designation for the virus responsible for AIDS.

More than 80% of patients diagnosed with AIDS or with the pre-AIDS syndrome are antibodies to HIV, and they have also been found very often in at-risk groups. There is great difficulty in diagnosing the risk of developing AIDS. It is known that AIDS develops in at least 10% of individuals infected with HIV, although it is. percentage is suspected to be higher.

Generally, a patient with AIDS is diagnosed when a previously healthy adult with an intact immune system acquires reduced T-cell immunity. Decreased immunity appears briefly over a period of 18 months to 3 years. As a result of this decreased immunity, patients become susceptible to opportunistic infections, to various types of cancer such as Kaposi's sarcoma and other disorders associated with reduced immune system functioning.

Another condition associated with HIV and the complex relationship with AIDS or ARC. This condition also leads, in some cases, to AIDS. There is currently no treatment capable of preventing disease or of reversing AIDS or ARC immunodeficiency. All patients with opportunistic infections and approximately half of all patients with Kaposi's sarcoma died within two years following diagnosis. Attempts to restore the immune system in AIDS patients have not been successful,

Recently, it has been reported that 3β-azido-3'-deoxy thymidine (AZT) is an antiviral agent that inhibits the infectivity and cytopathic effect of HIV-1 in vivo and prolongs life, (See Mitsuya, (1985) 7096-100, Fischl, et al., New Engl, J. Med.) However, preliminary results indicate that this compound exhibits toxicity and neurotoxicity in the bone marrow in a clinical trial. (See Yarchoan et al., Laneet (1986), 575-580) Richman, et al, AZT was first synthesized by Horwitz et al., J. Org. Chem., 29, 1964, In 1973, their activity against Friend's leukemia virus (a retrovirus) was reported (See Ostertag et al., Proc. Natl. Acad.

Know. USA, 71, (1974), 4980-49.85 and Krieg et al, Ex-pt1, Cell,

Res. 116, (1978), 21-29, and references cited therein),

The compounds of the present invention are not structurally related to AZT, U.S. Pat. 4,681,933 relates to certain 21,31-dideoxy-5-substituted-uridine and related compounds as antiviral agents. Structurally these compounds are AZT-like and are very different from the compounds of the present invention. ? 4,759,929 has particular relevance to the present application. In this patent, there is disclosed a method for the treatment of HIV-1 infection in warm-blooded animals which involves treatment with a salt of 9-antimony-III-21-tungs- NH4 +) - | This compound is a polyoxometalate, but from the point of view of the composition is different from the comounds of the present invention. However, there are drawbacks connected with this drug, such as: 1] induces thrombocytopenia and, in general, has a high toxicity on the cells of the human bone marrow and 2) should be administered intravenously, once degrades to a pH <4.

French Patent No. 2,587,214 relates to tungsten heteropolyions having an anticoagulant action for use in vivo and in vitro to prevent blood clotting,

Polyoxometalates or polyoxanools are condensed oligomeric aggregates of metal atoms and oxygen atoms occurring in two generic forms: those containing only these atoms (isopolyoxoanions, tsopolysaccharides or isopolyoxomatoses) and those containing one or more mats &quot; heteroatoms &quot; besides the metal and oxygen atoms (heteropolyions, heteropolysaccharides or heteropolyoxometalates). In this latter class of complements,

Further, the heteroatoms may be the major group ions such as 4+ 5+

Si (in the form of 1icotungstate = ST], P (as Dawon's tungstate, DT] or transition ion groups such as 3 + 2 + - - -

Fe or Co. Polyoxometalates have been known for a century but only recently has increased interest in these with stations. To a certain extent, this is due to the fact that these compounds are now chemically better defined. In addition, since 1977, polyoxometalates, and in particular heteropolymeric or heteropoly acids, have received increased attention as reagents or as catalysts for redox processes involving organic sub-treatments.

Despite known agents for the treatment of AIDS, there remains a need for new and effective methods for the treatment of retroviral infections, particularly HIV infections, including AIDS and the AIDS related complex (ARC). It was in this context that the present invention was realized,

Summary of the Invention

Accordingly, it is an object of the present invention to provide novel effective compositions for the treatment of viral infections, particularly for retrovirus infections; It is further an object of the present invention to provide methods for the treatment of viral infections, particularly for the treatment of retroviral infections;

It is also another object of the present invention to provide compounds and compositions for the treatment of AIDS or ARC in mammals, including humans.

These and other objects of the present invention, as will become more apparent later, were carried out by the discovery that the polyoxometalates with the above-identified structures exert inhibitory activity on the cells effected by various retroviruses, leading to an e. XDectively that these agents have in vivo activity against such retroviruses in mammals, such as humans,

BRIEF DESCRIPTION OF THE DRAWINGS The fuller appreciation of the invention and many of its advantages will be more readily achieved if the invention is well understood by reference to the following detailed description when taken in conjunction with the accompanying drawings, in which: Fig. 3 represents the relative effect of HPA-23, Fig. 3 shows the relative effect of HPA-23, Fig. 2 shows a synthetic scheme for ArgH-ST, the arginine salt of silicotungstate, Fig. ST, BT, NH4 BT and NH4 ST in the formation of human granulocyte-macrophage precursor cell colonies,

Detailed Description of the Preferred Aspects Polyoxometalates of the Invention

The polyoxometalates of the present invention may be represented by the following general formulas. -8 -8 n = 0-4 m = 0-5 n = 0-4 p = 0-6 (M + H) + (M + H) + = Wherein X represents a potassium or sodium atom or an ammonium group, histidine H, lysine H, arginine H, and the like; their hydrates can also represent any naturally occurring monocyclic amino acid or polypeptide (for example with 2 to 10 amino acids, preferably 2 to 4 amino acids containing a naturally occurring basic amino acid which may be arg, bis, lys its protonated form, therefore in the cationic form],

For the sake of simplicity and clarity the following abbreviations are used here: tStW12 ° 404 ° = ST (8Μ12 ° 40! 5 '= BT = W10 CP2W18 ° S2 ^ = DT hi strdina = Flts 1i sine = Lys arginine = Arg

Among the groups represented by X, for the present invention, the protonated basic amino acids of

The present invention relates to the use of natural occurrence, histidine H, lysine H and arginine H and, in particular, arginine H. However, other naturally occurring amino acids or small peptides, such as cations, can also be used for the purposes of the invention. forming a positive charge under acidic conditions. Examples of small peptides are Gly-Arg, Ala-β-Ala, and the like. Small peptides may not occur naturally, but will preferably be based on naturally occurring amino acids. Naturally occurring amino acids are generally chosen from among the 20 naturally occurring amino acids most commonly found in proteins,

The compounds of the present invention may be synthesized by conventional methods known in the art. Phosphotoxomethalates, such as W 1 O and FeT polyoxometalates such as ST, are most often prepared by careful acidification to correct concentrations of monomer tungstate, NaOH salt in aqueous solution with a water-soluble precursor of the heteroatom, if appropriate, followed by precipitation of the product, the appropriate water-soluble salt of the desired groups X + is added in the last step. In the Examples section, the processes for the preparation of S'T, BT and some of the salts of the ST and BT amino acids are provided.

Specific examples of these compounds which fall within the scope of the present invention are the following

Cnh41h3st Cnh4Ik4bt (; nh4 [2h2st cnh4j2h3ht

Cnh4! 4hbt

(NH4) 4ST

(NH4) 5BT (.UysH 1 n (Η) s_nBT, η = 1-5 kt (HlsH) n (H) 6.nDT, n = 1-6 Compounds of the general formula (Arg) (H) r BT in which n represents an integer of 1 to 5 to 5 are of particular interest.

When referring to antiviral activity in this invention is meant the ability of a compound to inhibit the development of an in vitro or in vivo virus. Viruses with particular interest in the present application are HIV-1 , HIV-2, herpes simplex virus type 1 (HSV-1) and Type 2 (JISV-2J and FMD-immunodeficiency virus), are of particular importance to HIV-1 and HIV-2, especially HIV-1. present compounds may also exhibit antiviral activity on other retroviruses and even on viruses in general such as herpes simplex virus types including herpes simplex virus type 1 and type 2 and the murine retrovirus (EY 10 and Cas-Br- The present compounds may furthermore exhibit antimicrobial properties.

Humans suffering from diseases caused, for example, by HIV, can be treated by administering to the disease a pharmaceutically effective amount of one or more of the compounds of the present invention, optionally, but preferably in the presence of a pharmaceutically acceptable carrier or solvent. Compatible pharmaceutically acceptable binding agents and / or adjuvants may also be included. The active ingredients may also be admixed with other active components which do not diminish the desired action and / or complement the desired action. The active substances according to the present invention may be administered by any route, for example o- parenteral, endovenous, intradermal, subcutaneous or topical, in solid form or in liquid form. For injection the medium used should be a sterile liquid. As the injectable medium it is preferred to use water containing stabilizing agents, solubilizers and / or conventional buffering agents in the case of injectable solutions.

Suitable additives include, for example, tartrate and borate buffers, ethanol, dimethyl sulfoxide, complexing agents (for example, ethylenediaminetetraacetic acid), high molecular weight polymers (for example liquid polyoxyethylene), for regulating viscosity or derivatives of polyethylene of sorbitan anhydrides.

Solids for vehicle use include, for example, starch, lactose, mannitol, methyl cellulose, talc, high dispersion salicylic acid, high molecular weight fatty acids (such as stearic acid), gelatin, agar calcium phosphate, magnesium stearate, animal and vegetable fats, or high molecular weight solids such as polyethylene glycols. Compositions suitable for oral administration may, if appropriate, contain flavoring agents and / or coloring agents. The preferred dosage form of the compounds of this invention is the oral route. Thus the compounds may be in the form of tablets.

The active substances according to the invention may be employed in dosages and amounts which are determined by conventional methods of pharmacology. Thus, for humans, the substances can be used in doses comprised between 0.1 and 100 mg / kg of body weight per day. More preferred limits range from 1 to 30 mg / kg body weight per day. More preferred limits are from 1 to 200 mg / kg of body weight per day. body and per day. The dosages may be administered once or divided into a number of smaller doses to be administered at varying time intervals.

It was expected that the compounds of the present invention will be useful in the treatment of mammals, including humans. Other mammals are dogs, cats, monkeys, horses and others.

To be useful, the compounds should exhibit relatively low toxicity to normal cells. The toxicities of some of the present compounds were evaluated in Vero cells and in human peripheral blood mononuclear cells and the toxicity values were determined. Toxicity tests are described below. The present invention, now generally described, is best understood by reference to certain specific examples which are included for the purpose of clarification only and should not be construed as limiting the present invention in any of its aspects, unless otherwise specified,

Synthesis of Representative Compounds i. H4stw12o4Q QJ, Pope, Michael Tfior,

Ffeteropoly and Tsopoly Oxometalates, Springer-Verlag,

Berlin, 1983, p. 1.

A solution of MgSOâ, "H2 O2 by solution of 25 g of the compound in 125 ml of deionized water on stirring was charged to a 250 ml round bottom flask. Thereafter, 1.25 ml of saturated aqueous sodium starch solution (NaSiO 3), (cooled water), was added, and the resulting mixture was heated at 80Â ° C for 5 minutes. At this point, was added 15 ml of concentrated hydrochloric acid for 1.5 hours using a dropping funnel to the hot solution under vacuum. The heating was then removed and transferred to a separatory funnel which The addition of 10 ml of hydrochloric acid and 50 ml of diethyl ether gave rise to three layers.The two upper layers were clear, the top layer being ethereal and the next layer There was then an ill-defined white-off separation zone of unknown composition probably composed of a mixture of the backsheet and the backsheet. The backscatter consisted of a clear viscous liquid which was the etherate contained the prod Each of the upper layers was removed and the ether layer was allowed to stand overnight at room temperature to form crystals. These crystals were recrystallized from water and the white crystalline product (14 g) was characterized by TV, VV and ¹³C NMR. Spectra were characteristic of the compound ΓΧ, Synthesis of Amino Acid Salts:

Lysinium, arginine and histidide from 'K A. Lysidinium salt (2j

This process was adapted from the E, O, North process for the sodium salt, Jasmine, C, et al. J. Natl, Cancer Inst. 53, (1974), 469. A saturated solution of 1 in water

3.6 g is used in a minimum amount of water. Thereafter, a saturated solution was prepared using 1 g of list monohydrochloride (Aldrich) in a minimal amount of water. The lysine solution was then added to the solution of 2, with stirring. The addition resulted in a Bright White precipitate whose polyoxymethalate nature was qualitatively confirmed by a rapid color change to blue when a small amount was exposed on a metal spatula for a few minutes. The precipitate was filtered under vacuum and a small amount dried under reduced pressure.

vacuum at 40 ° C. An IV of the dry substance 0:35) showed the presence of the characteristic peak set of as well as several peaks from a counter catton of lysine. Recrystallization of the compound was obtained by dissolving it in water with little heating and allowing the solution to stand at room temperature until the formation of crystals. After several days, blue-white gelatinous clods were formed. The clods were transformed into a white powder mass when filtered under vacuum using the same conditions of Recrystallization gave a second recrystallization of more lumps which became white microcrystals when filtered under vacuum, the product's ARMn confirmed the presence of lysine (by comparison with the yield spectrum). showed the presence of the characteristic peaks of lysine hydrochloride, as well as the peaks of references expected for the B. angin salt.

J

The same general procedure was The same preparation as that used for the preparation of 2 with the substitution of the arginine monoclortrate (Alfa) was used in the reverse reaction. In contrast to the lysine salt, a white precipitate only after the addition of 3 ml of the saturated solution, instead of forming immediately. Due to this formation of the slower precipitate, the solution was allowed to stir for 1 hour before under vacuum filtration to remove the product Recrystallization was carried out by treating 3 g of the compound with 25 ml of water.

Although the bulk of the compound remained insoluble after sonication and stirring under moderate heating, the filtrate was collected and allowed to stand at room temperature for several days. White crystals (0.5 g) were collected and collected , The IR spectrum of the product exhibited the characteristic peaks of the anion of 1, and the peaks from the opposite arginine cation were observed. NMR showed a peak profile almost identical to that observed for the initial cation of the opposite cation of arginine as well as adjusted to the spectrum of its components described in the literature.

In this preparation the same general procedure as in the present invention was used for the synthesis of 2 with substitution of the histidine hydrochloride CAlfaJ by the source of the opposite cation. A saturated solution of 1 was prepared by dissolving 4 g of A saturated solution of methylphosphine hydrochloride was added dropwise and the solution of 1% was added dropwise. During the initial additions, small particles of white precipitate were formed which disappeared rapidly by redissolution. As more cation solution was added, approximately 2 ml, this precipitate became permanent and after addition of 1 ml of solution, the resulting solution was allowed to stir for 1 hour and the precipitate formed from the filtrate The reaction was recrystallized from 1.71 g with 200 ml of water. Although some of the compound remained insoluble, the filtrate was collected and left rest until the formation of white microcrystals. The TV and the NMR of the compound were consistent with the components of the compound. ΓΠ - Synthesis of

The following was successively added to 100 ml of water under vigorous stirring: 50 g of 2.5 g of H2 O2 and 30 ml of 6M HCl. The resulting solution, mp about 6, was boiled for 8 hours . Water of time was added, when necessary. Any solid substance formed at this time was filtered off, dried (MgSOâ, ") and evaporated to dryness. the solution was acidified to pH2 with 6M hydrochloric acid and kept boiling for an additional 30 minutes. Precipitation was effected with 10 g of solid potassium chloride. After filtration and a wash with diethyl ether, the crude product was recrystallized at 60Â ° C in 25 ml of water. The infrared spectrum of the product was typical of the spectrum of the literature relating to the compound, IV, Synthesis of amino acid salts:

Lysidin, arginine and histidinium, from 5, A. Lysidinium salt (6):

The same general procedure was used for this synthesis as that used for the amino acid salts of A solution of 5 by dissolving 1.3 g in 25 ml of water was prepared. A cloudy liquid was obtained. A saturated solution of lysine hydrochloride in water was also prepared. This solution was added dropwise to solution 5 with stirring. During the addition the solution became progressively clear with the addition of each goat and the turbidity disappeared after the addition of 20 ml. The solution was allowed to stand to white to form rounded globules. Filtered and redissolved in water, allowing to recrystallize. After 10 days transparent polyhedral crystals were obtained. IR was used to characterize the compound. The IR spectrum indicated the presence of anion of 5 as well as the opposite cation of 11. The NMR spectrum showed the most conclusive proof of the presence of the lysine counter cation by comparing the spectrum with the spectrum of the lysine hydrochloride literature and with the spectrum of the starting compounds, B. Synthesis of a compound of formula

X is an NH4 +, Na + group,

Arginine H +, Histidine H +, from CD ·

These derivatives were prepared using a solution of the preparative phase before the precipitation of Nan by the addition of potassium hydroxide. Instead of potassium hydroxide, different salts were prepared by acidification of the solution with 6M hydrochloric acid The solution was then partitioned into equal aliquots, after which saturated aqueous solutions of the opposite cation were added until a pH of 2.30 was obtained. The solutions were allowed to stand for 30 minutes and the various precipitates were then filtered. 1. X represents arginine H + (7):

A solution of saturated aqueous arginine hydrochloride as the coupling source was added and 3.6 g of a white precipitate were obtained. This was characterized by IR and NMR spectra consistent with the components of the compound. 2, X represents fitstidine H + £ 81:

A saturated aqueous histidine hydrochloride solution was added as a source of cation, and 3.7 g of a white precipitate were obtained. The compound was characterized by IR and 1 H-NMR spectra consistent with the components of compound .

Biological Activity

At present, no animal experiments are available for HIV-1. Chimpanzees can be infected but they do not develop the disease. Thus, in vitro activity on infected cells is generally described in Cells, Human peripheral blood mononuclear cells [PBMC] from healthy donors seronegative to HIV-1 and hepatitis B viruses that were isolated by discontinuous Follic- -Hypaque, by centrifugation at 1:80 x g for 30 minutes, which were washed twice with phosphate buffered saline solution [pH 7.2, PBS] and agglomerated at 300 xg for 10 minutes, Prior to infection , the cells were stimulated with phyto hemagglutinin (PHA) at a concentration of 16.7 μg / ml for three days in RPMI 1640 medium supplemented with 15% warming inactivated fetal calf serum, 1.5 mM L-glutamine, 100 U / ml penicillin, 100æg / ml streptomycin and 4 mM sodium hydrogencarbonate buffer. Virus. LAV-1 strain HIV-1 virus was obtained by the delivery of Dr. P. Feonino (Centers for Disease Control, Atlanta, GA). These viruses were propagated in human PBMC using RPMI 1640 medium as previously described [McDougal, et al., J. Immun, Met. 76, (1985), 171-183, free of phytohemagglutinin and supplemented with Interleukin-2 97% v / v [Advanced Biotechnolgies, Silver Spring, M0], 7æg / ml DEAE-dextran [Pharmacia, Uppsala , Sweden), and 370 U / ml anti-human leukocyte [alpha]] leukocyte interferon (TCfi, Lisle, Π). Viruses were obtained from titrated cell-free culture supernatant and stored at -80 ° C until use,

Inhibition of Virus Replication in human PBMC.

Uniformly stimulated human PBMCs were uniformly distributed by uninfected PHAs in 25 cm flasks to give 5 ml of a suspension containing about 2 x 10 cells / ml. The virus was added in appropriate dilutions to infect the cultures. The mean inverse transcriptase (RT) activity of the inoculum was determined as 50,000 decompositions per minute / ml, corresponding to about 100 TCID q,, determined according to Gro opmanman et al., [Groopman J.E., et al., AIDS Res. Human Retro, -3 -21

., 71-857. The drugs were added to the final concentrations in duplicate in 5 ml of RPMI 1640 medium supplemented as previously described. Non-infected and untreated PBMCs were developed in parallel with controls at equal cell densities. Cultures were maintained in a humidified atmosphere with 5% carbon dioxide and 95% air, in incubator, at 37 ° C for six days after infection, at which time samples of all cultures were collected for analysis of the reverse transcriptase (RT) activity of the supernatant. Previous studies had indicated that maximum levels of RT were obtained at this time.

RT activity assay. 6 ml of supernatant from each culture was blotted out by centrifugation 300 x g for 10 minutes. The virus particles were pooled from 5 ml samples at the rate of 40,000 rpm for 30 minutes using a Beckman 70.1 Ti rotor and suspended in 200 ul virus fragmentation buffer (50 mM Tris-Cl, pH 7.8, 80% NaCl, 20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride and 0.5% Trfton X-100),

A reverse transcriptase assay was performed on 9.6 well microtiter plates according to the method of Spira et al., Spira, T., et al., J. In. The reaction mixture containing 50 mM Tris-Cl, pH 7.8, 9 mM MgCl2, 5 mM dithiothreitol (DTT), 4.7 μg / ml (rA) nt of the specific activity 78.0 Ci / mmole equivalent to 1730 cpm / pmole; NEN Research Products, Boston, MA, which was added to each well. The reaction mixture was then added, which was then incubated at 37øC for 2 hours. The reaction was quenched by the addition of 100æl of trichloroacetic acid at 10øC. % (TCA1 containing 0.45 mM sodium pyrophosphate,

The acid-insoluble nucleic acids precipitated having been collected on glass filters using the Skatron semi-automated harvesting device (set to 9). The filters were washed with 5% TCA and 70% ethanol, and placed in flashing ampoules,

4 ml scintillation liquid (Econofluor, NER 'Research Products, Boston, MA) was added and the amount of radioactivity in each sample was determined by using a Packard Tri-Carb liquid scintillation analyzer model 2,000CA, The results were expressed in dpm / ml of the original supernatant labeled.

Cttotoxicity test. The drugs were evaluated for their potential toxic effects on uninfected phytohemoglobin-stained human PBMCs. Seeds were seeded in the flasks so that the final cell concentration was 2 x 10 6 cells / ml, the cells were incubated with and without the drug for 6 days at a time when aliquots were removed to assess the viability of the cells assessed by the Trtpan blue dye exclusion method using a hemacytometre. The mean inhibitory concentration (IC q)) was calculated using the method of medium effect (Chou, J, and T.C. (1985), Esesefer-Biosoft, Elsevier Science Publishers, Cambridge, UK). Chou, T.C., and P. Talalay, Adv. Enz., Regulation 22 (1984) 27-55].

Evaluation of reverse-transcripts and reversed purified retrovirus. ΗΓV-1 reverse transcapeptase was isolated from detergent-disrupted viruses obtained from free supernatant from PBMC cells stimulated with infected phytohemagglutinin. The enzyme was purified by passages of the extract through ion exchange chromatography columns as described above (Eriksson, B, et al., Antimicrob.Chem.Chem 31, (1977) 600-604) Briefly, 750 ml of the virus-infected culture medium from virus-infected PBMC ultracentrifugation to agglomerate the virus. The pellet was dissolved in Tris-BCI buffer A5, pH 7.9 / 0.05% Nonidet P-40 / 5 mM dithiothreitol / 20% (w / v) glycerol containing 1 mM EDTA and frozen and thawed four times. After clarification by centrifugation, the enzyme was partially purified from the supernatant by passing the extract through a cellulose column / DEAE (Whatman DE-52, 2.6 x 15 cm [pre-equilibrated with buffer B (50 mM T.ris-Cl., PH 7.9, 50 mM KCl 1.1 mM EDTA, 1 mM dithiothreitol, glycerol at 20 °] .The elution was performed using a linear gradient 50-50% KCl in buffer B. The fractions containing the enzymatic activity buffer against buffer B and then purified on a Qfhatman P-11] phosphocellulose column using a linear gradient of KCl (50-500 mM) in buffer B. The peak fractions were pooled and dialyzed against buffer B. The enzyme was characterized as HIV-1 reverse transcriptase, based on its cation, salt, pH and matrix exogenies, according to tertiary descriptions (Eriksson, et al., Antimicrob. Agents Chemother. 31 (1977) 60Q-604). A standard reaction mixture was used for

to evaluate the inhibitory effect of the drugs as described above (Erifcsson, B., et al., Anti-Agrobacterial Agents, 31 (1997) 600-604). The reaction mixtures (final volume 100 μl) contained 100 mM Tri-HCl, pH 8.0, 50 mM KCl, 2 mM MgCl 2, 5 mM DTT, 400 μg / ml bovine serum albumin, 3 μg / ml (rA ) (1T) 1 Âμg of β] TTP (specific activity 82.3 Ci / mmol). The reactions were initiated by the addition of 10æl of partially purified reverse transcriptase and incubated at temperature of 37 ° C for 60 minutes are processed as described above (Eriksson, B., et al., Antimicrob.-Agents Chemother, 31 (1977) 600-604).

Cell culture assays for stromal virus vRNAs For the plaque assays, miRNA-free Vero cells obtained from the Flow LaBoratory (McLean, VA), plaque reduction methods and assays were used of cftotoxicity have been described previously (Schinazt, RF, et al., Anttrotcrob., Agents Chemo-ther, 22 Cl982). 499-507). Mean effect method - The dose-effect relationships were analyzed by the mean effect equation CCftot · »T.-C, and P. Talalay, Adv. Enz. Regul. 22, 984, 27-55). The values of the curve (m) and the mean effect and inhibitory concentration CCE50. and IC 50 were obtained through the use of a computer program developed by Chou and Chou CCRou &gt; etc. Chou, Elsevier-Biosoft, Elsevier Science Publisfiers, Cl985)? Cambrigde, UK),

UV / XC assay for Murine retrovTrus. Cells. SC-1 embryonic fibroblasts and XC cells were obtained from the American Type Culture Collection, Rockville, MD. The XC cell line was derived from a transplanted tumor induced in newborn White-Weston rats by intramuscular injection of the Prague strain of Rous sarcoma vTrus. This cell line does not release infectious virus. Cells were maintained in Eagle's minimal essential medium with Hanks's balanced salt solution, supplemented with 100 U / ml penicillin, 100æg / ml streptomycin, 24 mM sodium hydrogen carbonate, 25 mM Hepes and 2% (maintenance) or 10¾ (development) of inactivated fetal or newborn calf serum.

Quantification of vTrus by the UV / XC assay, XC test [Rowe, W, P., W. E. Pugh, and J. W. Hartley. &quot; Plaque assay techniques for murine leukemia viruses &quot;, Virology 42, (1970) 1136-1399) is a type of plaque virus that checks for the presence and type of murine leukemia virus B and N-tropic virus. Since these viruses do not cause obvious signs of infection on SC-1 mouse cells, it is necessary to coat the culture producing the virus with a cell line indicator, XC. The number of syncytia produced is a direct measure of the vTrus titre in FFU: - 5

Six wells were seeded with 1 X 10 SC-1 mouse cells on the first day. The next day the medium was removed and 0.2 ml of inoculum was added in medium containing polybrene (4æg / ml). For the determination of the antiviral activity of the compounds in the culture, the dilution of vTrus chosen produced in the untreated cultures about 80 foci per well. After 1 hour, vTrus were removed and the drug was added in various concentrations. Plates were filled with meto on the third and fifth days. The medium was removed on the eighth day and cells were exposed to UV radiation (60 erg / mm per second) for 30 seconds, the medium containing 10 XC cells was then added to each plate, the first tenth day, plaques were fixed with 10% buffered formalin and stained with crystal violet. Comparison of the number of foct with those found in cells infected by the non-drug-treated viruses allowed the determination of whether a drug in a The control cells, free of virus but receiving the drug, provided an estimate of the toxic effects of this, quantified by the use of the tripan blue calcium ion.

Bone Marrow Toxicity Test,

Preparation of Cells. Human bone marrow cells were collected by aspiration of the later iliac Christian from normal healthy volunteers treated with heparin and the mononuclear population was separated by Ficoll-Hypaque gradient centrifugation. Cells were washed twice with solution Hanks saline solution equilibrated, counted in a stoichiometer and 98% viability was obtained according to the trypan blue exclusion evaluation,

Pharmacological Cytotoxicity Assay · in macrophages,

Colony-Forming Formulations, the culture assay for CFU-GM was performed using the soft-agar double layer method as described recently by Sommadossi and Carltsle 'Antimtcrob,

Agents Chemotfier, 31, 0987) 452-454 A nutrient medium

McCoy 5A supplemented with 15% dialyzed fetal bovine serum (inactivated by heating at 56øC for 30 minutes) (Gibco Laboratories, Grand Tslands, NY) in all experiments. This medium was totally free of thymidine and uridine. Cells were cloned on 0.3% agar in the presence of increasing concentrations of the modulator compound or only in the meto (control).

After 14 days of incubation at 37øC in a humidified atmosphere containing 5% carbon dioxide in air, the colonies (> 50 cells) were counted using an inverted microscope.

Bone Marrow Toxicity Studies:

The toxicities and affinities of toxicities in these tests (results shown below) were not identical in the mouse toxicity studies. The toxicities and toxicity affinities in these tests (results given below) were not shown to be identical in the mouse toxicity studies. Bone marrow toxicity tests revealed results with some interest and very encouraging, 1] Ammonium assays proved to be substaminially less toxic than the free acid forms of any of the polyoxometalates.

2] The following order of toxicity was established in these assays: ammonium BT = NH4 + BT BT minus toxic] - BT and NH4 ST4 AZT and ST <tHPA-23, French polymetomeric action, -HIV-1) usual in the clinical trial (the toxic mats), Fig.

In fact, BT ammonium showed no toxicity to -28

The replacement of the inorganic cations of polyoxomethalates (Na, K and H, etc.) by ammonium and cations from basic amino acids does not undesirably affect the high antiviral efficacy and the low toxicity of the polyoxometalates displayed in cell cultures (PBMC), but these polyoxometalates exhibit a greatly improved toxicity to human bone marrow cells and markedly improved tolerance to the above salts,

The results of the activity analysis of some of the present compounds are shown in Table 1 below,

J 00 co 001 1 o r r '. coconut

J 0 CO 0 0 1 J 3 • Γ · x O c Q- 1 r- 0 fO to s. X cn S-CQ X 00 + - &gt; 1 3 0 00 co s: 0 < Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ

3 Ο ιηι Ο LU

00X

J CO ct5 fC + -> r- "O co C3 rC3 &lt; u &gt; S- (/)))) r r r r r 0 0 0 0 0. Φ)))) D D D D in in in in in in in in in in in. oo roo

X COO

CM

a &gt; X

(O-O-O-O-O-O-O-O-O-O-O-O- -Ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι 0 ι ι ι ι ι 0 0 0 0 0 0 0 0 0 0 0 0 0 ι ι ι ι ι ι ι ι ι ι ι ι ι ι Ο Ο ι Ο Ο Ο Ο Ο Ο Ο Ο Ο Ο Ο Ο Ο Ο Ο Ο Ο ------Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ-----Γ Γ Γ-Γ Γ Γ Γ Γ Γ Γ Γ Α Α Α Α - r «| | | | | | | | | ι ι ι ι ι ι ι ι ι ι ι ι ι 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 A CO fmmm r "~ OO CM CO co AAX- * OOAAAA to Γ-ν σι r- α LO LO A CM AAO LO CO CO rt- · CO CO cn-CM O Λ AAAAAAAAAO to CM OO r * x co CM X CM r- CM CO r to CO O CM X x- *. &lt; * »» x »/ -S,« - "x. OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO - 1-CO-lx. CM x-'' Sm-r-CO rx. r- "CM σι σι CO« 3 CM * 3 &quot; CO O A A A A A A A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0, A compound of the formula: ## STR1 ## wherein: (s) - (-) - S - N - CO 3 X 1 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 2 CO 1- a. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10,

J

the LOlo LU

J OO CO I I Ο Ο Π

X X

Γ '»CO CM C0 Λ Λ γ- CM O O υ X oo -C τ 1 1 sz α. S- or or <a ο p 2 2 cn 1 1 C / 7 -P-P co X α Ο < s: ο o oo LU Λ Pv. Ό Ό o o o o o o o o o o Ό Ό Ό Ό Ό Ό Ό Ό Ό 1 1 1 Ό Ό Ό 1 1 1 Ό Ό Ό Ό Ό Ό to to to to to to to to to

I &gt; · C / 7X I &lt; (0) Ί Ί Ί Ί Ί------------------Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί Ί T3 or

S-f0 3 O O) O ε o to CU a. C0 LO Λ Ο C0 Λ «V Ο 03

C01 ^. Λ CM LOΓ ΟΟ (NI CTi -λ Ο

PvCOo LOcr &gt; O

V CT &gt; cr "Λo

V oo COo X COo A co

p ^ LO CM &lt; υ or + - &gt; (Λ or O fO Ό CL • r- or ε u n3 -P or c to or D D 2 or Ξ CL CU ε h- 4- or M <D u_ CJ <

COcr &gt; HELLO

cr &gt; The

LO oo Ao cno CMooo COo CO 03 Ao

oo CM

CM to CM

OO CM A CM o

Hi, what's up?

C0 LO λ CM or • nÍ * 0)

CM LO 03o 00

CM OO LO OO ooΛ • ςί &quot; o Aco co A 00 Γ-Ν. how crazy

LO -H X + X LO> ->

«3 &quot; LO CM p ^ CMco

c • l

CO QC <= C LO LOAo CM CM ao

CO to CM CM

«= D

CO-

CO 1-4X

coo

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Loa

co LO <nP A OO

+ X with &gt; &gt; -

A O Oo

CM at &lt; vP

or r- * LO ACO as co + l + c <3 cc < HPA-23 (Compound-Reference) 0.29

J J

ο LO Ο HH /

or ί α; &gt; 2: â € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒS = K "D SI 2: x! - o • r * ta &gt; X 3. = *., 3 - 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 σ Cl O 0 0 0 0 ♦ r r r r r r r r X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 1 1 1 1 - CO co σ & AAA Λ AA Λ A Λ O (XI CO CO - - - ta ta ta ta ta ta ta ta ta ta ta ta ta ta ta Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ V =. 3 * = 3. X X 3 υ s: s ST X X S = N = * s X "O t" • r- O 3. ° O 3. 3 3 k 3. = v. = L ta; X o O O o O o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 'Ι Λ ΟΟ ΟΟ ΟΟ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ Λ ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι (1) (b) and (b) and (b). C)-C)-C)-C)-C CO-C CO-C 00-C 00-C 00-C 00-C 00-C 00-C --C --- -? +? O c Ccccc to -σ O cc: I 1 1 1 1 1 0 P l 1 ln LO LO ^ t * 3 - st Ol to LO ___- ____ E r - 0 - - + + + + + + O 3 O. &lt; / RTI &gt; -XXXXX CO., &Lt; / RTI &gt; -CQ 3 st -NH-. - &gt; - &gt; &Gt;. ✓-. IO or 1- to 3-amino. (1) where X is the number of atoms in which X is the number of atoms in which X is the number of atoms. χ a-X-X-X) KH> l · -1> · cu < M V V &gt; ftl to V v and 1 X as z X X S &lt; c X &lt; c a. &lt; 0 0 3 = Q 0 or Z '- - -' X

Assays in completely infected mice

Tests were performed on fully infected mice using one of the compounds of the present invention, the method of this test being identical to that used by Ruprecht et al., Nature 323, 467-469, with the exception of the use of vTrus Friend instead of vTrus Rauscher, The results of this test are shown in Table 2 below.

33 J

J U &quot; 'v 00 &lt; CQ tsr O JZ c tacn E 3 εεcu <Μ Μ Μ Μ '' '' 'O O O O! CL X LU &lt; 0 CMo E TJ ta 3 Cr

(1), and (2) and (2), and (2). Π3 CO co

CD co

CD CD (O δβ I * r- CO CO CO tm tm 1 1 «« «« «« «« «« «« «« «« «« «« «« «Ό Ό cn CM CM CO O in CO CO CO CO CO 1 + 1 + 1 + 1 1- +1 +1 + CO CO CO CO CO CO 1 + 1 + 1 + 1 - Ό Ό Ό «« 1 1 1 1 1 ο ο ο ο ο ο ο ο ο ο ο ο ο ο ο ο Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ the cn ca O p Μ o o o t t t t t Ι Ι Ι Ι Ι Ι Ι Ι Ι Ι Ι Ι «« «« «« «« « Γ-Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ ca ca ca ca ca ca ca ca ca ca ca ca. a Q Q o ta cn Ί- E cr θ cr θ 'cr θ cu' - ca Ό • p * 3 n- to ε CL · α. CL · a. Q-α. SL * a UI -σ c-t p-r f-c h-i F-1 M o o <c ε r- LO CU a. -3 tta CU 0) • &lt; r &gt; ui -PO Φ (CU EC cn -P T3 -ta ca * r- * CU ca T3 T3 T3 -a T3 T3 fO E E Ό -U -X -X -X LU ta 3 3 ε (1) and (2), and (3) is the same as that of the invention. -a "The CU CU" "UI" "" "" "" "" "" "" "" "" "" "" "" &gt; i. the &gt; • r- E cu 3 ·. TJ • P ta ta ε r h - - teu teu teu teu teu teu t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t. Ό &quot; + - &gt; ·································································· (c) (c) &gt; &lt; u • &gt; X &lt; / RTI &gt; C C NJ M J0 a CL a a · · co a. Τ 't- &lt; &lt; &lt; X-1. mkd: mg / kg / d

-34-

Taking into account the teachings above, it is obvious that numerous modifications and variations of the present invention are possible. It is to be understood, therefore, that the present invention may be embodied in other forms than those specifically described within the scope of the appended claims.

Claims (7)

(H) BW, n 5-n 12 40 or - ** + 4 * (basic amino acid) Wherein n is an integer of from 1 to 5 or from 1 to 4, respectively, characterized in that the compound of formula (I) carefully acidifying a monomeric tungstate at the correct concentrations using, generally, as the starting material the sodium salt, NaW03, in aqueous solution, with a water-soluble heteroatom precursor; the resulting product is subsequently precipitated and the appropriate water soluble salt of the desired cations is added at the last stage of the synthesis. 2.- 2. A process according to claim 1, wherein the basic amino acid is selected from arginine, lysine and histidine. 3. A process for the preparation of pharmaceutical compositions for oral or parenteral administration suitable for the treatment of infections caused by retroviruses such as HIV-1 or HIV-2 in a mammal characterized in that an effective amount , from 0.1 to 100 mg / kg, of a compound of the general molecular formula chosen from &lt; XVH + WSiW12 ° 4 <n = 0-4, (X +) n (H +> 5-m BW12 ° 40> 5 'm = 0-5, <X +) nHH + WW10O32> 4 ~ n = 0-4, (X +) pHH +) 6-p2P18W18 ° 62 &gt; 6 'Ό II 0 1 σ &gt; in which X represents a potassium or sodium atom, an NH4 + group, a natural monoolethonic amino acid or a polypeptide consisting of natural amino acids and bearing a natural basic amino acid selected from arginine, lysine and histidine in the protonated form, n is zero or an integer from 1 to 4, m represents zero or an integer from 1 to 5, and p represents zero or an integer from 1 to 6 with a pharmaceutically acceptable carrier. 4.- 4. A compound according to claim 3, wherein a compound of general formula (Arg H +) n <H +) 5n (BW 12 O 40) 5 wherein n is an integer from 1 to 5. 5. A compound as claimed in claim 3, wherein a compound of formula B '(H +) 5 - n' is wherein n is an integer from 3 to 5. 6. A process according to claim 3, wherein the monocarboxylic species is selected from a cation K +, Na +, NH4 +, arginine H +, lysine H +, histidine H + or of formula NR2 + in which R represents a alkyl group. 7. A method for the treatment of infections such as AIDS or ARC, caused by retroviruses such as HIV-1 or HIV-2, in a mammal, including man, for administering to said mammal via oral or parenteral composition, an effective amount, comprised between 0.1 and 100 mg / kg body weight / day, preferably between 1 and 30 mg / kg body weight / day, and more preferably between 1 and 20 mg / kg total body weight / day, of a compound of formula mole- (X +) 5-mTBW12 ° 40) 5-m = 0-5, (X +) n -H-NMR (DMSO-d6) (X +) â € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒ (X +) â € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒ (X +) n (H +) 4-n (w + a potassium or sodium atom, an NH4 + group, a naturally occurring monocyclic amino acid or a polypeptide consisting of natural amino acids and having a natural basic amino acid selected from arginine, lysine and histidine in the protonated form, is zero or an integer of? to 4, m is zero or an integer from 1 to 5, P is zero or an integer from 1 to 6. The Portuguese Patent Office